AbstractStaphylococcus aureus nuclease (SNase) homologues, previously thought to be restricted to bacteria and archaea, are demonstrated by sequence analysis to be present also in eukary‐otes. The human cellular coactivator pi00 is shown to contain four repeats, each of which is a SNase homologue. Surprisingly, these repeats are unlikely to possess SNase‐like activities as each lacks equivalent SNase catalytic residues, yet they may mediate plOO's single‐stranded DNA‐binding function. Products of Corydalis sem‐pervirens and Saccharomyces cerevisiae open reading frames are predicted to adopt the same fold and possess similar functions as SNase. Five additional hypothetical proteins of bacterial origin are also predicted to be active SNase‐like nucleases, including one that appears to be C‐terminally truncated in a manner analogous to an engineered active SNase variant. Conservation of Asp‐19 and Asp‐83 among these homologues suggests a re‐evaluation of the roles of these residues in Ca2+‐binding and/or catalysis.